金頂側耳中免疫調節蛋白的純化與活性評估

Purification of Immunomodulatory Protein from Pleurotus citrinopileatus and Evaluation of its Bioactivity

王壐凱(Hsi-Kai Wang)

指導教授 ： 許輔

共同指導教授 ： 許明仁

國立臺灣大學/生物資源暨農學院/園藝學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02494

摘要

本研究新發現金頂側耳 (Pleurotus citrinopileatus Sing.)含有免疫調節蛋白P. citrinopileatus protein (PCiP)。金頂側耳子實體經均質、超音波破碎、飽和硫酸銨沈澱、透析後，再以 DE-52, Hi-trap Q離子交換層析純化出之免疫調節蛋白PCiP，其分子量約為 15 kDa。以等電聚膠分析(isoelectric focusing, IEF) 測得PCiP之等電點pI值約為5.2。醣蛋白染色試驗發現PCiP不為醣蛋白。血液凝集試驗發現PCiP對小鼠紅血球並無凝集作用。免疫調節活性試驗發現5 ~ 200 μg/mL之PCiP可直接刺激RAW264.7巨噬細胞分泌TNF-α及nitric oxide；對於小鼠脾細胞而言，PCiP於高濃度時可活化細胞激素IFN-γ的分泌；於低濃度時可促進小鼠脾細胞的增生活性；5 ~ 160 μg/mL 之PCiP可抑制由concanavalin A (ConA) 所促進之細胞增生活性。細胞毒性試驗發現PCiP並無細胞毒性。這些結果說明金頂側耳免疫調節蛋白PCiP對於刺激巨噬細胞活化上具有免疫調節活性，在臨床藥學上具有應用發展之潛力。

關鍵字

純化 ； 金頂側耳 ； 免疫調節 ； 蛋白質

並列摘要

A new immunomodulatory proteins, PCiP, was purified from the fruiting bodies of Pleurotus citrinopileatus Sing. The isolation procedure of PCiP includes homogenization, sonication, precipitation by ammonium sulfate, and followed by DE-52 ion-exchange chromatography. The proteins from P. citrinopileatus can be further purified using FPLC with Hi-trap Q anion-exchange chromatography. SDS-PAGE analyses showed that the molecular mass of PCiP was about 15 kDa. Further staining using the periodic acid/Schiff reagents showed that PCiP was not a glycoprotein. Isoelectric focusing electrophoresis reveals that the isoelectric point (pI) of PCiP was approximately 5.2. Additionally, PCiP could not agglutinate mouse red blood cells. The immunomodulatory activities of PCiP was demonstrated by their potent stimulatory activity toward mouse splenocytes and RAW 264.7 macrophages. PCiP (5~200 μg/mL) could directly activate the cells and enhance the production of nitric oxide and TNF-

並列關鍵字

Purification ； Immunomodulatory ； Pleurotus citrinopileatus ； Protein

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